Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling NR2F2 with ab211777 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mesenchymal cells of human tonsil tissue (PMID: 11026559) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Lane 1 : Anti-TRIM24 antibody [EPR22825-2] (ab256491) at 1/1000 dilution
Lane 2 : Anti-NR2F2 antibody [EPR18443] (ab211777) at 1/1000 dilution

Lane 1 : Wild-type HCT116 cell lysate
Lane 2 : NR2F2 knockout HCT117 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 46 kDa
Observed band size: 45 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab211777 observed at 45 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab211777 was shown to react with NR2F2 in wild-type HCT116 cells in western blot. The band observed in knockout cell line ab266888 (knockout cell lysate ab257186) lane below 45kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HCT116 and NR2F2 knockout HCT117 cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab211777 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-NR2F2 antibody [EPR18443] (ab211777) at 1/1000 dilution

Lane 1 : MCF7 (human breast adenocarcinoma cell line) whole cell lysate
Lane 2 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : Human fetal kidney tissue lysate
Lane 4 : HEK-293 (human epithelial cell line from embryonic kidney) whole cell lysate
Lane 5 : K562 (human chronic myelogenous leukemia cell line from bone marrow ) whole cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
Lanes 1-2 & 4-5 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Lane 3 : Goat Anti-Rabbit IgG Peroxidase Conjugate, specific to the non-reduced form of IgG at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 46 kDa
Observed band size: 46 kDa


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM /TBST 

All lanes : Anti-NR2F2 antibody [EPR18443] (ab211777) at 1/1000 dilution

Lane 1 : C6 (rat glial tumor cell line) whole cell lysate
Lane 2 : PC-12 (rat adrenal gland pheochromocytoma cell line) whole cell lysate
Lane 3 : NIH/3T3 (mouse embryo fibroblast cell line) whole cell lysate
Lane 4 : Rat brain tissue lysate
Lane 5 : Rat spleen tissue lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 46 kDa
Observed band size: 46 kDa


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM /TBST 

Anti-NR2F2 antibody [EPR18443] (ab211777) at 1/1000 dilution + Human NR2F1 full length recombinant protein (negative control) at 10 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Developed using the ECL technique.

Predicted band size: 46 kDa


Exposure time: 3 minutes

Blocking and dilution buffer: 5% NFDM/TBST

Human NR2F1 full length recombinant protein containing aa1-423 with a His-tag was made in house.

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling NR2F2 with ab211777 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mesenchymal cells of human testis tissue (PMID: 11026559) is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human fetal spleen tissue labeling NR2F2 with ab211777 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mesenchymal cells of human fetal spleen tissue is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labeling NR2F2 with ab211777 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on tumor cells of human breast cancer tissue is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded human colon cancer tissue labeling NR2F2 with ab211777 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on tumor cells of human colon cancer tissue is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse liver tissue labeling NR2F2 with ab211777 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mesenchymal cells of mouse liver tissue is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat lung tissue labeling NR2F2 with ab211777 at 1/250 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mesenchymal cells of rat lung tissue is observed. Counter stained with hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling NR2F2 with ab211777 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line.

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab211777 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) preadsorbed (ab150120) secondary at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HEK-293 (human epithelial cell line from embryonic kidney) cells labeling NR2F2 with ab211777 at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HEK293 cell line.

The nuclear counterstain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) (ab150120) secondary antibody at 1/1000 dilution (red).

The negative controls are as follows:
-ve control 1: ab211777 at 1/100 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed (ab150120) secondary at 1/1000 dilution.
-ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary at 1/1000 dilution.

NR2F2 was immunoprecipitated from 1 mg of MCF7 (human breast adenocarcinoma cell line) whole cell lysate with ab211777 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab211777 at 1/500 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1: MCF7 whole cell lysate 10 μg (Input).
Lane 2: ab211777 IP in MCF7 whole cell lysate.
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab211777 in MCF7 whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 1 second.